US11174389B2 - Phosphole compound - Google Patents

Phosphole compound Download PDF

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US11174389B2
US11174389B2 US16/329,151 US201716329151A US11174389B2 US 11174389 B2 US11174389 B2 US 11174389B2 US 201716329151 A US201716329151 A US 201716329151A US 11174389 B2 US11174389 B2 US 11174389B2
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optionally substituted
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phosphole
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US20190264031A1 (en
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Shigehiro Yamaguchi
Aiko NAKA
Masayasu Taki
Chenguang Wang
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Nagoya University NUC
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    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B57/00—Other synthetic dyes of known constitution
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
    • C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6564—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having phosphorus atoms, with or without nitrogen, oxygen, sulfur, selenium or tellurium atoms, as ring hetero atoms
    • C07F9/6568—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having phosphorus atoms, with or without nitrogen, oxygen, sulfur, selenium or tellurium atoms, as ring hetero atoms having phosphorus atoms as the only ring hetero atoms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks

Definitions

  • the present invention relates to a phosphole compound.
  • Fluorescent organic compounds that have high fluorescence quantum yields are important as luminescent materials for organic EL elements or as fluorescent dyes for in vivo fluorescence imaging. There have been numerous reports on both basic research and applied research of fluorescent organic compounds.
  • phosphole compounds having a specific structure are also known as fluorescent dyes (see, for example, Patent Literature (PTL) 1 and Non-patent Literature (NPL) 1).
  • PTL Patent Literature
  • NPL Non-patent Literature
  • the phosphole compound disclosed in PTL 1 can maintain a high fluorescence quantum yield in any solvent ranging from low-polarity to high-polarity solvents, and some embodiments of the phosphole compound disclosed in PTL 1 are fluorescent dyes with excellent light resistance.
  • the fluorescent dyes disclosed in PTL 1 and NPL 1 have high fluorescence quantum yields and excellent light resistance. There is, however, still room for improvement in terms of fluorescence quantum yield in environments containing large amounts of water. For example, cells, tissues, living organisms, etc. are mainly composed of water. The object to be observed by fluorescence bioimaging is present in a trace amount and is very small. Therefore, water solubility and a high fluorescence quantum yield of the fluorescent dye in aqueous solutions are important for high-sensitivity observation with a high signal-to-noise ratio.
  • the development of a molecule capable of efficiently emitting fluorescence even in water has been desired for use as a fluorophore for fluorescence bioimaging of cells, tissues, living organisms, etc.
  • the fluorescent dyes disclosed in NPL 1 and PTL 1 do not dissolve in water. Furthermore, the fluorescence quantum yield of the fluorescent dye disclosed in PTL 1 is reduced when water is added to the organic solvent.
  • the fluorescent dye disclosed in NPL 1 has an absorption peak wavelength of 367 nm, and thus cannot be excited with such lasers of short wavelengths. Therefore, application of such light sources to the fluorescent dye disclosed in NPL 1 is difficult. Further, in consideration of phototoxicity against cells, using a longer wavelength laser is preferable.
  • An object of the present invention is to provide a fluorescent dye that is capable of maintaining a high fluorescence quantum yield irrespective of solvent polarity, and providing an improved fluorescence quantum yield and light resistance even in environments containing large amounts of water, and that is also widely applicable for fluorescence bioimaging of cells, tissues, living organisms, etc.
  • the present inventors conducted extensive research and found that phosphole compounds having a specific structure have a high fluorescence quantum yield irrespective of solvent polarity, achieve an improved fluorescence quantum yield even in environments containing large amounts of water, and have significantly improved light resistance, as compared with conventional fluorescent dyes; and that therefore, such phosphole compounds are fluorescent dyes that can withstand repeated super-resolution microscopy observation, such as stimulated emission depletion (STED) imaging.
  • STED stimulated emission depletion
  • Ar 1 and Ar 2 are the same or different, and represent an optionally substituted aromatic hydrocarbon ring or an optionally substituted heteroaromatic ring;
  • Ar 3 represents a divalent ⁇ -conjugated unit;
  • R 1 represents an optionally substituted alkyl group, an optionally substituted cycloalkyl group, an optionally substituted aryl group, or an optionally substituted heteroaryl group;
  • R 2 and R 3 are the same or different, and represent a hydrogen atom, an optionally substituted alkyl group, an optionally substituted cycloalkyl group, an optionally substituted aryl group, or an optionally substituted heteroaryl group; and
  • Z represents a reactive group).
  • Item 7 A fluorescent dye comprising the phosphole compound according to any one of Items 1 to 6.
  • Item 8 The fluorescent dye according to Item 7, which is for stimulated emission depletion (STED) imaging.
  • Item 9. A protein labeling agent comprising the phosphole compound according to Item 5 or 6.
  • Item 10. A stimulated emission depletion (STED) imaging method using the phosphole compound according to any one of Items 1 to 6 or the fluorescent dye according to Item 7 or 8.
  • a protein labeling kit comprising the phosphole compound according to Item 5 or 6, the fluorescent dye according to Item 7 or 8, or the protein labeling agent according to Item 9.
  • a protein labeling method comprising reacting a protein with the phosphole compound according to Item 5 or 6, the fluorescent dye according to Item 7 or 8, or the protein labeling agent according to Item 9.
  • the phosphole compound of the present invention has an absorption peak in the visible light wavelength range (in particular, about 400 to 500 nm) irrespective of solvent polarity, and has a high fluorescence quantum yield. Furthermore, the phosphole compound of the present invention can have enhanced fluorescence quantum yield even in environments containing large amounts of water, and is widely applicable to fluorescence bioimaging of cells, tissues, living organisms, etc.
  • the phosphole compound of the present invention is a fluorescent dye suitable for repeated super-resolution microscopy observation, such as stimulated emission depletion (STED) imaging.
  • the phosphole compound of the present invention wherein the reactive group represented by Z is an amine reactive group or a thiol reactive group can function as a protein labeling agent that labels proteins.
  • FIG. 1 shows Kohn-Sham plots and HOMO and LUMO energy levels of Phox 430 NHS Ester obtained in Example 6 and C-Naphox obtained in Comparative Example 3.
  • FIG. 2 shows UV-visible absorption and fluorescence spectra of Phox 430 NHS Ester in various solvents.
  • FIG. 3 is graphs showing fluorescence quantum yields of Phox 430 NHS Ester of Example 6 and C-Naphox of Comparative Example 3 in mixed solvents of DMSO and HEPES that are mixed at various ratios.
  • the solution concentrations were adjusted to the optical density (Alexa Fluor 488: 0.22, C-Naphox: 0.22, PB430: 0.21) at an excitation wavelength of 460 nm.
  • FIG. 6 is a graph showing pH-dependence of fluorescence of Phox-COOH (10 ⁇ M) obtained in Example 8.
  • FIG. 8 shows the results of confocal imaging and STED imaging of immunofluorescently labeled vimentin.
  • FIG. 8( a ) is a confocal fluorescence microscopy image of tubulin immunolabeled with Phox-NHS Ester (Example 9)
  • FIG. 8( b ) is a STED microscopy image of tubulin immunolabeled with Phox-NHS Ester (Example 9)
  • FIG. 8( c ) is a graph showing optical resolutions of the corresponding confocal (orange line) and STED (crimson line) microscopy images.
  • FIG. 9 shows the photostability of Alexa Fluor 488 (Comparative Example 4) and Phox-NHS Ester (Example 9) under STED conditions.
  • FIG. 9( a ) is STED microscopy images of tubulin immunolabeled with Alexa Fluor 488 repeatedly captured 5 times (Comparative Example 4);
  • FIG. 9( b ) is STED microscopy images of tubulin immunolabeled with Phox-NHS Ester (Example 9) repeatedly captured 5 times;
  • FIG. 9( c ) is normalized intracellular fluorescence intensity plotted as a function of the number of recorded STED microscopy images.
  • FIG. 10( a ) is a confocal fluorescence microscopy image of tubulin immunolabeled with Phox-NHS Ester (Example 9).
  • FIG. 10( b ) is a Z-scan STED microscopy image of tubulin immunolabeled with Phox-NHS Ester (Example 9) at a depth of 2 ⁇ m.
  • FIG. 10( c ) shows a 3D structure of tubulin immunolabeled with Phox-NHS Ester (Example 9).
  • FIG. 11( a ) is a confocal fluorescence microscopy image of tubulin immunolabeled with Alexa Fluor 488 (Comparative Example 4).
  • FIG. 11( b ) is a Z-scan STED microscopy image of tubulin immunolabeled with Alexa Fluor 488 (Comparative Example 4) at a depth of 2 ⁇ m.
  • FIG. 11( c ) shows a 3D structure of tubulin immunolabeled with Alexa Fluor 488 (Comparative Example 4).
  • FIG. 12( a ) is a confocal microscopy image of microtubules immunolabeled with Phox-COOH in a fixed HeLa cell, with an insert of an enlarged view of the selected region.
  • FIG. 12( b ) shows a line profile taken along the arrow in FIG. 13( a ) across the filaments.
  • FIG. 13 is fluorescence images of immunolabeled microtubules in a fixed HeLa cell.
  • FIG. 13( a ) is a confocal microscopy image (left), a STED microscopy image (middle), and intensity profiles (right).
  • the confocal and STED microscopy images each include an enlarged view of the portion surrounded by dotted lines.
  • the intensity profiles show the confocal microscopy image with a black line, and the STED microscopy image with a green line.
  • FIG. 13( b ) shows the first five STED microscopy images stained with Phox-COOH.
  • FIG. 13( c ) shows the first five STED microscopy images stained with Alexa Fluor 488.
  • 13( d ) shows integrated fluorescence intensity plotted as a function of the number of recorded STED images. All images were recorded with excitation at 470 nm, and a STED laser of 592 nm (CW-STED, 30 mW) was used for STED. Scare bars indicate 2 ⁇ m.
  • FIG. 14( a ) shows repeatedly captured STED microscopy images. Each numeral represents the number of flames.
  • FIG. 14( b ) shows typical intensity profiles of microtubules labeled with Phox-COOH in the images (number of flames: 1, 10, 20 and 30). The full width at half maximum (FWHM) was computed at a resolution of microtubules calculated from the Gaussian fit.
  • FIG. 14( c ) shows statistical analysis of FWHM at 10 fluorescence spots using the Gaussian fit.
  • FIG. 15( a ) is a three-dimensional STED image of HeLa cell microtubules immunolabeled with Phox-COOH, including a color scale corresponding to the height (increments in z-direction: 50 nm, scale bar: 5 ⁇ m).
  • FIG. 15( b ) shows STED images along the corresponding xz and yz planes in FIG. 15( a ) .
  • FIG. 16 shows a comparison of z-scan STED images of microtubules immunolabeled with ( a ) Alexa Fluor 488, ( b ) Alexa Fluor 430, ( c ) Star 440SXP, ( d ) Atto 425, and ( e ) Phox-COOH (PB430).
  • Each numeral indicates the number of recorded z-scan images.
  • Z-scan imaging was performed in steps of 50 nm and with excitation at 470 nm and a fluorescence depletion laser of 592 nm (CW-STED, 30 mW; STED 3D z donut, 50%). Scale bars indicate 2 ⁇ m.
  • FIG. 17( a ) to FIG. 17( e ) show a comparison of fluorescence images of immunolabeled microtubules repeatedly photographed while being irradiated with a 470-nm confocal laser (WLL, 12 ⁇ W), the microtubules having been immunolabeled with ( a ) Alexa Fluor 430, ( b ) STAR 440SX, ( c ) Alexa Fluor 488, ( d ) Atto 425, or ( e ) Phox-COOH (PB430). Each numeral indicates the number of recorded confocal images. Scale bar: 2 ⁇ m.
  • FIG. 17( f ) shows integration fluorescence intensities (I) relative to the initial value (I 0 ) plotted as a function of the number of recorded images.
  • FIG. 17( g ) shows first-order plots based on changes in fluorescence intensity.
  • the photobleaching rate was calculated from the slope of the straight line and normalized to that of Alexa Fluor 430.
  • the relative photostability refers to the reciprocal of the photobleaching rate normalized in Table 2.
  • FIG. 18 shows a comparison of photostability between Alexa Fluor 430 (( a ) to ( c )) and Phox-COOH (PB430) (( d ) to ( f )).
  • First and second STED images of Alexa Fluor 430-labeled vimentin (( a ), ( b )) and Phox-COOH (PB430)-labeled tubulin (( d ), ( e )) were consecutively photographed (scale bar: 2 ⁇ m), and their relative fluorescence intensities were compared (( c ), ( f )).
  • FIG. 19 shows a two-color imaging scheme combined with STED microscopy based on the distinctly different photostability of Phox-COOH (PB430) and Alexa Fluor 430. Microtubules and vimentin filaments were separately immunolabeled with Phox-COOH (PB430) and Alexa Fluor 430, respectively. Two STED microscopy images (deconvoluted data) were recorded consecutively with excitation at 470 nm (WLL, 10 ⁇ W) and fluorescence depletion at 592 nm (CW-STED, 30 mW).
  • FIG. 19( c ) is an image obtained by subtracting (removing) the image of FIG. 19( b ) from the image of FIG. 19( a ) .
  • FIG. 19( d ) is a two-color STED microscopy image obtained by combining the image of FIG. 19( b ) with the image of FIG. 19( c ) .
  • FIG. 20 shows the determination of two-color STED imaging resolution based on different photostability.
  • FIG. 20( a ) is a two-color STED microscopy image (deconvoluted data) of microtubules (green) and vimentin filaments (magenta) separately immunolabeled with Phox-COOH (PB430) and Alexa Fluor 430, respectively.
  • the intensity profile plotted along the dotted lines was intersected with microtubules ( b ) and vimentin filaments ( d ) in the inset of the two-color STED image. The profile was fit to Gaussian distribution.
  • the phosphole compound of the present invention is a compound represented by formula (1):
  • Ar 1 and Ar 2 are the same or different, and represent an optionally substituted aromatic hydrocarbon ring or an optionally substituted heteroaromatic ring;
  • Ar 3 represents a divalent ⁇ -conjugated unit;
  • R 1 represents an optionally substituted alkyl group, an optionally substituted cycloalkyl group, an optionally substituted aryl group, or an optionally substituted heteroaryl group;
  • R 2 and R 3 are the same or different, and represent a hydrogen atom, an optionally substituted alkyl group, an optionally substituted cycloalkyl group, an optionally substituted aryl group, or an optionally substituted heteroaryl group;
  • Z represents a reactive group.
  • the phosphole compound of the present invention which has a fused phosphole skeleton, has excellent light resistance and can have various reactive groups introduced thereinto via Ar 3 . Due to the presence of Ar 3 as a mediator in this manner, the phosphole compound can exhibit a high fluorescence quantum yield even in environments containing large amounts of water.
  • the terminal reactive group various substituents can be introduced.
  • an amine reactive group, a thiol reactive group, or the like is introduced, the resulting phosphole compound can be used as a protein labeling agent (in particular, an antibody labeling agent) that labels a protein (in particular, an antibody).
  • the phosphole compound of the present invention can inhibit a decrease in fluorescence brightness during repeated super-resolution microscopy observation, such as in vivo stimulated emission depletion (STED) imaging, the phosphole compound of the present invention is suitable for use in repeated super-resolution microscopy observation, such as in vivo stimulated emission depletion (STED) imaging.
  • Examples of the aromatic hydrocarbon ring represented by Ar 1 in formula (1) include monocyclic aromatic hydrocarbon rings and polycyclic aromatic hydrocarbon rings.
  • Examples of the monocyclic aromatic hydrocarbon ring include a benzene ring.
  • Examples of the polycyclic aromatic hydrocarbon ring include a naphthalene ring, an anthracene ring, a phenanthrene ring, a fluorene ring, a pyrene ring, a triphenylene ring, and the like.
  • the aromatic hydrocarbon ring represented by Ar 1 may optionally have one or more substituents.
  • substituents include alkyl groups described below, cycloalkyl groups described below, aryl groups described below, heteroaryl groups described below, alkenyl groups (e.g., vinyl, propenyl), alkynyl groups (e.g., ethynyl, 1-propynyl group), carbonyl, cyano, nitro, and the like.
  • the number of substituents is, for example, preferably 1 to 6, and more preferably 1 to 3.
  • Examples of the heteroaromatic ring represented by Ar 1 in formula (1) include a pyrrolidine ring, a pyrrole ring, a tetrahydrothiophene ring, a thiophene ring, an oxorane ring, a furan ring, an imidazole ring, a pyrazole ring, a thiazole ring, an oxazole ring, a piperidine ring, a pyridine ring, a pyrazine ring, an indole ring, an isoindole ring, a benzimidazole ring, a quinoline ring, an isoquinoline ring, a quinoxaline ring, and the like.
  • the heteroaromatic ring represented by Ar 1 may optionally have one or more substituents.
  • substituents include alkyl groups described below, cycloalkyl groups described below, aryl groups described below, heteroaryl groups described below, alkenyl groups (e.g., vinyl, propenyl), alkynyl groups (e.g., ethynyl, 1-propynyl), carbonyl, cyano, nitro, halogen atoms (e.g., fluorine, chlorine, bromine, iodine), and the like.
  • the number of substituents is, for example, preferably 1 to 6, and more preferably 1 to 3.
  • Ar 1 is preferably an optionally substituted aromatic hydrocarbon ring, and more preferably an optionally substituted polycyclic aromatic hydrocarbon ring.
  • the phosphole compound of the present invention can be either a phosphole compound represented by formula (1A):
  • Examples of the aromatic hydrocarbon ring represented by Ar 4 in formulas (1A) and (1B) include those described above.
  • the kind and number of substituents can also be the same as described above.
  • those represented by formula (1B) are preferable from the viewpoint of decreasing the energy difference between the HOMO level (highest unoccupied molecular orbital energy level) and the LUMO level (lowest unoccupied molecular orbital energy level) and increasing the absorption peak wavelength and the fluorescence peak wavelength.
  • Examples of the aromatic hydrocarbon ring represented by Ar 2 in formula (1) may be the same as described above. The same applies to the kind and number of substituents.
  • heteroaromatic ring represented by Ar 2 in formula (1) may be the same as described above. The same applies to the kind and number of substituents.
  • Ar 2 include optionally substituted aromatic hydrocarbon rings. Particularly preferable examples are unsubstituted aromatic hydrocarbon rings.
  • Examples of the divalent ⁇ -conjugated unit represented by Ar 3 in formula (1) include alkenylene, alkynylene, arylene, heteroarylene, and like groups.
  • alkenylene group examples include vinylene, propenylene, butenylene, and like C 2-6 alkenylene groups, in particular, C 2-4 alkenylene groups.
  • the alkenylene group may optionally have one or more substituents.
  • substituents include cycloalkyl groups described below, aryl groups described below, heteroaryl groups described below, alkenyl groups (e.g., vinyl, propenyl), alkynyl groups (e.g., ethynyl, 1-propynyl), alkoxy groups, carbonyl, cyano, nitro, halogen atoms (e.g., fluorine, chlorine, bromine, iodine), and the like.
  • the alkenylene group has one or more substituents, the number of substituents may be, for example, preferably 1 to 6, and more preferably 1 to 3.
  • the alkoxy group as a substituent means a group represented by —OR.
  • R in the alkoxy group includes not only alkyl groups described below, but also groups having alkyl chains linked to each other by an ether bond via an oxygen atom, groups having a carboxy group bonded to an alkyl chain, groups having alkyl chains linked to each other by an ether bond via —COO—, and the like.
  • Specific examples include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, —O((CH 2 ) p O) q CH 3 (wherein p is an integer of 1 to 5, in particular, an integer of 1 to 3; and q is an integer of 1 to 20, in particular, an integer of 2 to 10), —O—CH 2 —COOH, —O—CH 2 —COOC 2 H 5 , —O((CH 2 ) r —COO) s CH 3 (wherein r is an integer of 1 to 5, in particular, an integer of 1 to 3; and s is an integer of 1 to 20, in particular, an integer of 2 to 10), and the like.
  • alkynylene group examples include ethynylene, propynylene, butynylene, and like C 2-6 alkynylene groups, in particular, C 2-4 alkynylene groups.
  • the alkynylene group may have one or more substituents.
  • substituents include cycloalkyl groups described below, aryl groups described below, heteroaryl groups described below, alkenyl groups (e.g., vinyl, propenyl), alkynyl groups (e.g., ethynyl, 1-propynyl), alkoxy groups described above, carbonyl, cyano, nitro, and the like.
  • the alkynylene group has one or more substituents, the number of substituents is, for example, preferably 1 to 6, and more preferably 1 to 3.
  • arylene group examples include phenylene, naphtylene, anthracenylene, phenanthrenylene, fluorenylene, pyrenylene, triphenylene, and the like.
  • the arylene group may have one or more substituents.
  • substituents include alkyl groups described below, cycloalkyl groups described below, aryl groups described below, heteroaryl groups described below, alkenyl groups (e.g., vinyl, propenyl), alkynyl groups (e.g., ethynyl, 1-propynyl), alkoxy groups described above, carbonyl, cyano, nitro, halogen atoms (e.g., fluorine, chlorine, bromine, iodine), and the like.
  • the number of substituents is, for example, preferably 1 to 6, and more preferably 1 to 3.
  • heteroarylene group examples include pyrrolidylene, pyrolylene, tetrahydrothienylene, thienylene, oxolanylene, furanylene, imidazolene, pyrazolene, thiazolene, oxazolene, piperidinylene, pyridylene, pyrazylene, indolylene, cindolylene, benzimidazolylene, quinolylene, isoquinolylene, quinoxalylene, and the like.
  • the heteroarylene group may optionally have one or more substituents.
  • substituents include alkyl groups described below, cycloalkyl groups described below, aryl groups described below, heteroaryl groups described below, alkenyl groups (e.g., vinyl, propenyl), alkynyl groups (e.g., ethynyl, 1-propynyl group), alkoxy groups described above, carbonyl, cyano, nitro, halogen atoms (e.g., fluorine, chlorine, bromine, iodine), and the like.
  • the number of substituents is, for example, preferably 1 to 6, and more preferably 1 to 3.
  • Ar 3 is preferably an optionally substituted arylene group.
  • Ar 3 is more preferably an unsubstituted arylene group, and even more preferably phenylene.
  • Ar 3 is most preferably m-phenylene from the viewpoint of introducing a protein-labeling site, while optical properties (e.g., absorption peak wavelength, fluorescence peak wavelength, fluorescence quantum yield) of the dye are less affected.
  • Examples of the alkyl group represented by R 1 in formula (1) include both straight-chain alkyl groups and branched-chain alkyl groups. Examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, and like C 1-10 alkyl groups, in particular, C 1-6 alkyl groups.
  • the alkyl group represented by R 1 may have one or more substituents.
  • substituents include cycloalkyl groups described below, aryl groups described below, heteroaryl groups described below, alkenyl groups (e.g., vinyl, propenyl), alkynyl groups (e.g., ethynyl, 1-propynyl), alkoxy groups described above, carbonyl, cyano, nitro, halogen atoms (e.g., fluorine, chlorine, bromine, iodine), and the like.
  • the number of substituents is, for example, preferably 1 to 6, and more preferably 1 to 3.
  • Examples of the cycloalkyl group represented by R 1 in formula (1) include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and like C 3-10 cycloalkyl groups, and in particular, C 4-8 cycloalkyl groups.
  • the cycloalkyl group represented by R 1 may have one or more substituents.
  • substituents include alkyl groups described above, cycloalkyl groups described above, aryl groups described below, heteroaryl groups described below, alkenyl groups (e.g., vinyl, propenyl), alkynyl groups (e.g., ethynyl, 1-propynyl), alkoxy groups described above, carbonyl, cyano, nitro, halogen atoms (e.g., fluorine, chlorine, bromine, iodine), and the like.
  • the number of substituents is, for example, preferably 1 to 6, and more preferably 1 to 3.
  • Examples of the aryl group represented by R 1 in formula (1) include both monocyclic aryl groups and polycyclic aryl groups. Examples include C 6-18 aryl groups, in particular, C 6-14 aryl groups. Examples of monocyclic aryl groups include phenyl. Examples of polycyclic aryl groups include naphthyl, anthracenyl, phenanthrenyl, biphenyl, terphenyl, fluorenyl, pyrenyl, triphenylenyl, and the like.
  • the aryl group represented by R 1 may have one or more substituents.
  • substituents include alkyl groups described above, cycloalkyl groups described above, aryl groups described above, heteroaryl groups described below, alkenyl groups (e.g., vinyl, propenyl), alkynyl groups (e.g., ethynyl, 1-propynyl), alkoxy groups described above, carbonyl, cyano, nitro, halogen atoms (e.g., fluorine, chlorine, bromine, iodine), and the like.
  • the number of substituents is, for example, preferably 1 to 6, and more preferably 1 to 3.
  • heteroaryl group represented by R 1 in formula (1) examples include pyrrolidinyl, pyrrolyl, tetrahydrothienyl, thienyl, oxolanyl, furanyl, imidazolyl, pyrazolyl, thiazolyl, oxazolyl, piperidyl, pyridyl, pyrazyl, indolyl, isoindolyl, benzimidazolyl, quinolyl, isoquinolyl, quinoxalyl, and the like.
  • the heteroaryl group represented by R 1 may optionally have one or more substituents.
  • substituents include alkyl groups described above, cycloalkyl groups described above, aryl groups described above, heteroaryl groups described above, alkenyl groups (e.g., vinyl, propenyl), alkynyl groups (e.g., ethynyl, 1-propynyl), alkoxy groups described above, carbonyl, cyano, nitro, halogen atoms (e.g., fluorine, chlorine, bromine, iodine), and the like.
  • the number of substituents is, for example, preferably 1 to 6, and more preferably 1 to 3.
  • R 1 is preferably an optionally substituted aryl group, more preferably an optionally substituted phenyl group, and still more preferably phenyl from the viewpoint of the absorption peak wavelength and the fluorescence peak wavelength.
  • alkyl, cycloalkyl, aryl, and heteroaryl groups represented by R 2 and R 3 in formula (1) may be the same as described above. The same applies to the kind and number of substituents.
  • R 2 and R 3 are preferably optionally substituted aryl groups.
  • substituted aryl groups are more preferable, aryl groups substituted with alkoxy are even more preferable, aryl groups substituted with, for example, —O((CH 2 ) p O) g CH 3 (wherein p is an integer of 1 to 5, and in particular, an integer of 1 to 3; and q is an integer of 1 to 20, and in particular, an integer of 2 to 10) or —O(CH 2 ) r SO 3 H (wherein r is an integer of 1 to 5, and in particular, an integer of 1 to 3) are particularly preferable.
  • Aryl groups substituted with —O(CH 2 ) r SO 3 H (wherein r represents an integer of 1 to 5, and in particular, an integer of 1 to 3) are the most preferable.
  • the reactive group represented by Z in formula (1) is not particularly limited.
  • the phosphole compound wherein Z is carboxy; alkoxycarbonyl; hydroxy; amino; haloganated alkyl, such as chloromethyl; isocyano; isothiacyano; or the like can be easily converted to have a protein labeling group (e.g., an amine reactive group, a thiol reactive group) at position Z by reacting the reactive group with a compound having a desired substituent.
  • a protein labeling group e.g., an amine reactive group, a thiol reactive group
  • a group of compounds wherein Z is carboxy or alkoxycarbonyl, or a protein labeling group falls within the scope of the phosphole compound of the present invention.
  • the amine reactive group refers to a group that is reactive with an optionally substituted amino group possessed by the compound to be labeled.
  • a protein in particular, an antibody
  • the phosphole compound of the present invention can function as a protein labeling agent (in particular, an antibody labeling agent).
  • the thiol reactive group is a group that is reactive with an optionally substituted thiol group possessed by the compound to be labeled.
  • the phosphole compound of the present invention can function as a protein labeling agent (particularly an antibody labeling agent) by reacting with an optionally substituted thiol group possessed by a protein (in particular, an antibody).
  • amine reactive groups or thiol reactive groups are groups terminally having one of the following structures represented by formulas (2A) to (2E) (i.e., an amine-reactive end or a thiol-reactive end):
  • R 4 represents a hydrogen atom or a sulfo group
  • R 5 represents an optionally substituted alkyl group
  • the bond indicated by a solid line and a dashed line represents a single bond or a double bond
  • alkyl group represented by R 5 in formula (2C) may be the same as described above. The same applies to the kind and number of substituents.
  • the amine reactive group or thiol reactive group that satisfies such conditions preferably has an amine reactive end or a thiol reactive end via a linking group, such as a group represented by —O—, —COO—, or —CONR 6 — (wherein R 6 represents a hydrogen atom or an alkyl group described above) (in particular, —COO—).
  • a linking group such as a group represented by —O—, —COO—, or —CONR 6 — (wherein R 6 represents a hydrogen atom or an alkyl group described above) (in particular, —COO—).
  • n is an integer of 1 to 6
  • n is preferably an integer of 1 to 6, and more preferably an integer of 1 to 4.
  • the amine reactive group is preferably
  • the thiol reactive group is preferably
  • Ar 2a represents an optionally substituted aromatic hydrocarbon ring
  • Ar 3a represents an optionally substituted arylene group
  • R 1a represents an optionally substituted aryl group
  • R 2a and R 3a are the same or different and each represent aryl substituted with alkoxy
  • Ar 4 is as defined above
  • phosphole compounds described in the Examples below are even more preferable.
  • the phosphole compound of the present invention may be a hydrate or a solvate of the phosphole compound represented by formula (1).
  • the hydrate and solvate are both included within the scope of the present invention.
  • the method for producing the phosphole compound of the present invention is not particularly limited.
  • R 2a and R 3a are the same or different and each represent an aryl group substituted with a group represented by —OR 9 (wherein R 9 represents an alkyl group described above, in particular, methyl in consideration of ease of performing subsequent steps); R 2 and R 3b are the same or different and each represent a hydroxy-substituted aryl group; R 2c , R 3c and R 8c are the same or different and each represent an aryl group substituted with —O((CH 2 ) p O) q CH 3 or —O(CH) r SO 3 H (wherein p, q, and r are as defined above).
  • alkyl groups represented by R 8 and R 9 in Reaction Schemes 1 and 2 may be the same as described above. The same applies to the kind and number of substituents.
  • halogen atoms represented by X 1 in Reaction Schemes 1 and 2 include chlorine, bromine, iodine, and the like.
  • the protective group at the end of compound (3) or compound (3A) is preferably haloganated with a halogenating agent.
  • halogenating agent examples include iodine (I 2 ), iodine monochloride (ICl), iodine trichloride (ICl 3 ), N-iodosuccinimide (NIS), bromine (Br 2 ), N-bromosuccinimide (NBS), chlorine (Cl 2 ), and the like.
  • the amount of halogenating agent to be used is preferably 1 to 5 moles (in particular, 1.5 to 3 moles) per mole of the compound (3) or compound (3A).
  • the reaction can be usually performed in the presence of a reaction solvent.
  • the reaction solvents include aliphatic halogenated hydrocarbons such as dichloromethane, dichloroethane, chloroform, and carbon tetrachloride; amide solvents such as dimethylformamide; and the like. From the viewpoint of ease of synthesis, yield, etc., halogenated hydrocarbons are preferable, and dichloromethane is more preferable.
  • These reaction solvents can be used singly or in a combination of two or more.
  • the reaction atmosphere can be an inert gas atmosphere (argon gas atmosphere, nitrogen gas atmosphere, etc.).
  • the reaction can be performed with heating, at ordinary temperature, or with cooling.
  • the reaction temperature is preferably ⁇ 50 to 100° C., and more preferably 0 to 50° C.
  • the reaction time is not particularly limited and is preferably a period during which the reaction sufficiently proceeds.
  • a purification process can also be performed by a usual method, if necessary.
  • the subsequent step can be performed without performing a purification process.
  • step (2-1) compound (4) or compound (4A) obtained in step (2-1) and a compound represented by formula (8):
  • Examples of the boronic acid or the boronic acid ester group represented by R 10 in formula (8) include groups represented by formula (9):
  • Examples of the alkyl group represented by R 11 in formula (9) may be the same as described above. The same applies to the kind and number of substituents.
  • Examples of the boronic acid or the boronic acid ester group represented by R 10 include, for example,
  • the amount of compound (8) to be used is preferably 1 to 5 moles (in particular, 1.5 to 3 moles) per mole of compound (4) or compound (4A).
  • a palladium catalyst that is usually used in Suzuki-Miyaura coupling.
  • Specific examples include palladium acetate (Pd(OAc) 2 ), tetrakis(triphenylphosphine) palladium (0) (Pd(PPh 3 ) 4 ), palladium trifluoroacetate, palladium chloride, palladium bromide, palladium iodide, tris(dibenzylieneacetone)dipalladium (0) (Pd 2 (dba) 3 ), and the like.
  • tetrakis(triphenylphosphine)palladium (0) (Pd(PPh 3 ) 4 ) is preferable.
  • the amount of palladium catalyst to be used is preferably 0.01 to 0.3 moles, and more preferably 0.02 to 0.1 moles, per mole of compound (4) or compound (4A).
  • a ligand compound can also be used, if necessary.
  • a ligand compound usually used in Suzuki-Miyaura coupling can be used in an amount usually used.
  • a base can be used, if necessary.
  • the base include potassium fluoride, cesium fluoride, sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, cesium carbonate, potassium phosphate, sodium acetate, potassium acetate, calcium acetate, and the like.
  • potassium phosphate is preferable from the viewpoint of yield and ease of synthesis. From the viewpoint of ease of synthesis, yield, etc., the amount of base to be used is preferably 1 to 20 moles, and more preferably 3 to 10 moles, per mole of compound (4) or compound (4A).
  • reaction can usually be performed in the presence of a reaction solvent.
  • reaction solvents include ethers, such as diethyl ether, diisopropyl ether, tetrahydrofuran (THF), 1,4-dioxane, dimethoxyethane (DME), diglyme, cyclopentylmethyl ether (CPME), tert-butyl methyl ether (TBME), and anisole; aromatic hydrocarbons, such as benzene, toluene, and xylene; aliphatic halogenated hydrocarbons, such as dichloromethane, dichloroethane, chloroform, and carbon tetrachloride; nitrile solvents, such as acetonitrile; amide solvents, such as dimethylformamide; and the like.
  • ethers such as diethyl ether, diisopropyl ether, tetrahydrofuran (THF), 1,4-dioxane, dim
  • reaction solvents can be used singly or in a combination of two or more.
  • a mixed solvent of such an organic solvent and water can also be used.
  • the reaction atmosphere can be an inert gas atmosphere (argon gas atmosphere, nitrogen gas atmosphere, etc.).
  • the reaction can be performed with heating, at ordinary temperature, or with cooling.
  • the reaction temperature is preferably 0 to 150° C., and more preferably 50 to 100° C.
  • the reaction time is not particularly limited and is preferably a period during which the reaction sufficiently proceeds.
  • a purification process can also be performed by a usual method, if necessary.
  • the subsequent step can be performed without performing a purification process.
  • compound (5) or compound (5A) is hydrolyzed with an acid catalyst to obtain compound (6) or compound (6A).
  • the acid catalyst to be used for hydrolysis may be, for example, an acid commonly used for hydrolysis of ester.
  • R 8 is a less reactive group, such as t-butyl
  • a strong acid such as trifluoroacetic acid or hydrochloric acid, is preferably used. Since the acid catalyst is usually a liquid, the acid catalyst is preferably used in an excess amount.
  • reaction solvents include ethers, such as diethyl ether, diisopropyl ether, tetrahydrofuran (THF), 1,4-dioxane, dimethoxyethane (DME), diglyme, cyclopentylmethyl ether (CPME), tert-butyl methyl ether (TBME), and anisole; aromatic hydrocarbons, such as benzene, toluene, and xylene; aliphatic halogenated hydrocarbons, such as dichloromethane, dichloroethane, chloroform, and carbon tetrachloride; nitrile solvents, such as acetonitrile; amide solvents, such as dimethylformamide; and the like.
  • ethers such as diethyl ether, diisopropyl ether, tetrahydrofuran (THF), 1,4-dioxane, dimethoxyethane (DME), diglyme, cycl
  • reaction solvents can be used singly or in a combination of two or more.
  • the reaction atmosphere can be an inert gas atmosphere (argon gas atmosphere, nitrogen gas atmosphere, etc.).
  • the reaction can be performed with heating, at ordinary temperature, or with cooling.
  • the reaction temperature is preferably ⁇ 50 to 100° C., and more preferably 0 to 50° C.
  • the reaction time is not particularly limited and is preferably a period during which the reaction sufficiently proceeds.
  • a purification process can also be performed by a usual method, if necessary.
  • the subsequent step can be performed without performing a purification process.
  • compound (6B) can be obtained by dealkylating compound (6A) with a Lewis acid.
  • Lewis acids examples include boron tribromide, aluminum tribromide, boron trichloride, aluminum trichloride, titanium tetrachloride, tin tetrachloride, boron trifluoride, iodotrimethyl silane, silicon tetrachloride, and the like. These Lewis acids can be used singly or in a combination of two or more. Since Lewis acid is usually a liquid, Lewis acid is preferably used in an excess amount relative to the amount of compound (6A).
  • reaction can be usually performed in the presence of a reaction solvent.
  • reaction solvents include aromatic hydrocarbons, such as benzene, toluene, and xylene; aliphatic halogenated hydrocarbons, such as dichloromethane, dichloroethane, chloroform, and carbon tetrachloride; nitrile solvents, such as acetonitrile; amide solvents, such as dimethylformamide; and the like. From the viewpoint of, for example, ease of synthesis and yield, aliphatic halogenated hydrocarbons are preferable, and dichloromethane is more preferable. These reaction solvents can be used singly or in a combination of two or more.
  • the reaction atmosphere can be an inert gas atmosphere (e.g., argon gas atmosphere, nitrogen gas atmosphere).
  • the reaction can be performed with heating, at ordinary temperature, or with cooling.
  • the reaction temperature is preferably ⁇ 50 to 100° C., and more preferably 0 to 50° C.
  • the reaction time is not particularly limited and is preferably a period during which the reaction sufficiently proceeds.
  • a purification process can also be performed by a usual method, if necessary.
  • the subsequent step can be performed without performing a purification process.
  • compound (6B) is reacted with a compound represented by formula (10A): R 12 O((CH 2 ) p O) q CH 3 (10A) (wherein p and q are as defined above, and R 12 represents tosyl), or a compound represented by formula (10B):
  • step (2-6) described below can be skipped.
  • Examples of the alkylene group represented by R 13 in formula (10B) include C 1-6 (in particular, C 2-4 ) alkylene groups. Examples include methylene, ethylene, trimethylene, tetramethylene, and the like. The alkylene group may have one or more substituents.
  • substituents include, but are not limited to, alkyl groups described above, cycloalkyl groups described above, aryl groups described above, heteroaryl groups described above, alkenyl groups (e.g., vinyl, propenyl), alkynyl groups (e.g., ethynyl, 1-propynyl), alkoxy groups described above, carbonyl, cyano, nitro, halogen atoms (e.g., fluorine, chlorine, bromine, iodine), and the like.
  • the alkylene group has one or more substituents, the number of substituents is preferably, but not limited to, 1 to 6, and more preferably 1 to 3.
  • the amount of compound (10A) or compound (10B) is preferably 2 to 20 moles, and more preferably 3 to 10 moles, per mole of compound (6B).
  • a base can also be used, if necessary.
  • bases include ammonium chloride, potassium fluoride, cesium fluoride, sodium hydroxide, potassium hydroxide, sodium methoxide, sodium hydrogen carbonate, potassium hydrogen carbonate, sodium carbonate, potassium carbonate, cesium carbonate, potassium phosphate, sodium acetate, potassium acetate, calcium acetate, and the like. These bases can be used singly or in a combination of two or more.
  • potassium carbonate is preferable from the viewpoint of yield and ease of synthesis.
  • the amount of base to be used is preferably 3 to 50 moles, and more preferably 5 to 20 moles, per mole of compound (6B), from the viewpoint of ease of synthesis, yield, etc.
  • the reaction can be usually performed in the presence of a reaction solvent.
  • the reaction solvent include ethers, such as diethyl ether, diisopropyl ether, tetrahydrofuran (THF), 1,4-dioxane, dimethoxyethane (DME), diglyme, cyclopentylmethyl ether (CPME), tert-butyl methyl ether (TBME), and anisole; aromatic hydrocarbons, such as benzene, toluene, and xylene; aliphatic halogenated hydrocarbons, such as dichloromethane, dichloroethane, chloroform, and carbon tetrachloride; nitrile solvents such as acetonitrile; amide solvents, such as dimethylformamide; and the like. From the viewpoint of ease of synthesis, yield, etc., nitrile solvents are preferable, and acetonitrile is more preferable. These reaction solvents can be used singly or
  • the reaction atmosphere can be an inert gas atmosphere (argon gas atmosphere, nitrogen gas atmosphere, etc.).
  • the reaction can be performed with heating, at ordinary temperature, or with cooling. It is usually more preferable that the reaction is performed under reflux.
  • the reaction time is not particularly limited and is preferably a period during which the reaction sufficiently proceeds.
  • a purification process can also be performed by a usual method, if necessary.
  • the subsequent step can be performed without performing a purification process.
  • compound (7) is hydrolyzed using a base catalyst to obtain compound (6C).
  • the base catalyst to be used for the hydrolysis may be a base catalyst commonly used in hydrolysis of ester. Lithium hydroxide is preferable. Since the base catalyst is usually a liquid, the base catalyst is preferably used in an excess amount.
  • reaction solvents include ethers, such as diethyl ether, diisopropyl ether, tetrahydrofuran (THF), 1,4-dioxane, dimethoxyethane (DME), diglyme, cyclopentyl methyl ether (CPME), tert-butyl methyl ether (TBME), and anisole; aromatic hydrocarbons, such as benzene, toluene, and xylene; aliphatic halogenated hydrocarbons, such as dichloromethane, dichloroethane, chloroform, and carbon tetrachloride; nitrile solvents, such as acetonitrile; amide solvents, such as dimethylformamide; and the like. From the viewpoint of ease of synthesis, yield, etc., ether is preferable, and tetrahydrofuran is more preferable. These reaction solvents can be used singly
  • the reaction atmosphere can be an inert gas atmosphere (argon gas atmosphere, nitrogen gas atmosphere, etc.).
  • the reaction can be performed with heating, at ordinary temperature, or with cooling.
  • the reaction temperature is preferably ⁇ 50 to 100° C., and more preferably 0 to 50° C.
  • the reaction time is not particularly limited and is preferably a period during which the reaction sufficiently proceeds.
  • a purification process can also be performed by a usual method, if necessary.
  • the subsequent step can be performed without performing a purification process.
  • the carboxy group of compound (6) or compound (6C) can be replaced with a desired reactive group by a usual method.
  • a condensing agent can be used.
  • usable condensing agents include carbodiimide condensing agents (N,N′-dicyclohexylcarbodiimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and diisopropylcarbodiimide), imidazole condensing agents (carbonyldiimidazole and 2-chloro-1,3-dimethylimidazolinium chloride), triazine condensing agents (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride), phosphonium condensing agents (benzotriazol-1-yloxy-trisdimethylaminophosphonium salts, (benzotriazol-1-yloxy
  • a phosphole compound of the present invention having a succinimide skeleton (compound (1C) or compound (1C1)) at the end can be obtained by reacting N-hydroxysuccinimide, TSTU, etc.
  • the thus obtained phosphole compound of the present invention having a succinimide skeleton at the end can function as a protein labeling agent (in particular, an antibody labeling agent).
  • the amount of condensing agent to be used is preferably 1 to 5 moles, and more preferably 1.5 to 3 moles, per mole of compound (6) or compound (6C).
  • a carbodiimide reagent is preferably used as a coupling reagent.
  • carbodiimide reagents include dicyclohexylcarbodiimide (DCC), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), and the like. These carbodiimide reagents can be used singly or in a combination of two or more. These usable carbodiimide reagents may be, for example, in the form of hydrochloride or like salts (e.g., 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI)).
  • the amount of coupling reagent to be used is preferably 1 to 5 moles, and more preferably 1.5 to 3 moles, per mole of compound (6) or compound (6C).
  • a base is preferably used.
  • bases include pyridine, dialkylaminopyridine (e.g., 4-dimethylaminopyridine (DMAP)), and the like.
  • DMAP 4-dimethylaminopyridine
  • Such bases can be used singly or in a combination of two or more.
  • the amount of base to be used is preferably 1 to 5 moles, and more preferably 1.5 to 3 moles, per mole of compound (6) or compound (6C).
  • reaction solvents examples include ethers, such as diethyl ether, diisopropyl ether, tetrahydrofuran (THF), 1,4-dioxane, dimethoxyethane (DME), diglyme, cyclopentylmethyl ether (CPME), tert-butyl methyl ether (TBME), and anisole; aromatic hydrocarbons, such as benzene, toluene, and xylene; aliphatic halogenated hydrocarbons, such as dichloromethane, dichloroethane, chloroform, and carbon tetrachloride; nitrile solvents, such as acetonitrile; amide solvents, such as dimethylformamide; and the like. From the viewpoint of ease of synthesis, yield, etc., amide solvents are preferable, and dimethylformamide is more preferable. These reaction solvents can be used singly or in a
  • the reaction atmosphere can be an inert gas atmosphere (argon gas atmosphere, nitrogen gas atmosphere, etc.).
  • the reaction can be performed with heating, at ordinary temperature, or with cooling.
  • the reaction temperature is preferably ⁇ 50 to 100° C., and more preferably 0 to 50° C.
  • the reaction time is not particularly limited and is preferably a period during which the reaction sufficiently proceeds.
  • this phosphole compound can be reacted with a maleimide compound, such as N-(2-aminoethyl) maleimide, to thereby obtain a phosphole compound of the present invention having a maleimide skeleton at the end.
  • a maleimide compound such as N-(2-aminoethyl) maleimide
  • the maleimide compound to be used may be in the form of a salt, such as trifluoroacetate.
  • the amount of maleimide compound to be used is preferably 0.2 to 5 moles, and more preferably 0.5 to 2 moles, per mole of compound (1C) or compound (1C1).
  • a base is preferably used.
  • usable bases include pyridine, dialkylaminopyridines (e.g., 4-dimethylaminopyridine (DMAP)), amines (e.g., triethylamine), and the like. These bases can be used singly or in a combination of two or more.
  • the base is preferably used in an excess amount per mole of compound (1C) or compound (1C1).
  • reaction solvents include ethers, such as diethyl ether, diisopropyl ether, tetrahydrofuran (THF), 1,4-dioxane, dimethoxyethane (DME), diglyme, cyclopentylmethyl ether (CPME), tert-butyl methyl ether (TBME), and anisole; aromatic hydrocarbons, such as benzene, toluene, and xylene; aliphatic halogenated hydrocarbons, such as dichloromethane, dichloroethane, chloroform, and carbon tetrachloride; nitrile solvents, such as acetonitrile; amide solvents, such as dimethylformamide; dimethyl sulfoxide (DMSO); and the like. From the viewpoint of ease of synthesis, yield, etc., dimethyl sulfoxide is preferable. These solvents can be used singly
  • the reaction atmosphere can be an inert gas atmosphere (argon gas atmosphere, nitrogen gas atmosphere, etc.).
  • the reaction can be performed with heating, at ordinary temperature, or with cooling.
  • the reaction temperature is preferably ⁇ 50 to 100° C., and more preferably 0 to 50° C.
  • the reaction time is not particularly limited and is preferably a period during which the reaction sufficiently proceeds.
  • the fluorescent dye of the present invention comprises the phosphole compound of the present invention described above.
  • the fluorescent dye of the present invention which has a fused phosphole skeleton, has excellent light resistance (photostablity) and can have various reactive groups (Z) introduced via Ar 3 . Further, due to Ar 3 functioning as a mediator in this manner, the fluorescent dye of the present invention can have a high fluorescence quantum yield even in environments containing large amounts of water.
  • the fluorescent dye of the present invention is suitable for repeated super-resolution microscopy (in particular, stimulated emission depletion (STED) microscopy) observation, such as in vivo stimulated emission depletion (STED) imaging.
  • target proteins include avidin, streptavidin, annexin V, anti-IgG antibody, anti-IgM antibody, anti-CD3 antibody, anti-CD4 antibody, anti-CD20 antibody, anti-CD25 antibody, anti-CD43 antibody, anti-CD44 antibody, anti-CD68 antibody, anti-IFN- ⁇ antibody, anti-TNF- ⁇ antibody, anti-Ly-6G antibody, anti-Ku70 antibody, anti-IL-4 antibody, anti-IL-17 antibody, anti-IL-31 antibody, anti-Notch1 antibody, anti-Notch3 antibody, anti-FOXBP3 antibody, anti-Ki-67 antibody, anti-HLA-A2 antibody, anti- ⁇ -tubulin antibody, anti-cathepsin D antibody, anti-angiotensin antibody, anti-COX1 antibody, anti-GLUT1 antibody, anti-AKT1/2/3 antibody, anti-Apg3
  • the protein labeling agent (in particular, an antibody labeling agent) of the present invention comprises the phosphole compound of the present invention and is preferably in the form of a solution obtained by dissolving the phosphole compound in an organic solvent.
  • the content of the phosphole compound of the present invention is preferably 1 ⁇ 10 ⁇ 8 to 1 ⁇ 10 ⁇ 4 mol/L, and more preferably 1 ⁇ 10 ⁇ 7 to 1 ⁇ 10 ⁇ 5 mol/L.
  • fluorescent dye (phosphole compound) of the present invention is used as a protein labeling agent (in particular, an antibody labeling agent) in the form of a solution
  • examples of usable organic solvents include, but are not limited to, both polar solvents and nonpolar solvents.
  • polar solvents examples include ether compounds (tetrahydrofuran, anisole, 1,4-dioxane, cyclopentyl methyl ether, etc.), alcohols (methanol, ethanol, allyl alcohol, etc.), ester compounds (ethyl acetate etc.), ketones (acetone etc.), halogenated hydrocarbons (dichloromethane, chloroform, etc.), dimethyl sulfoxide, amide solvents (N,N-dimethylformamide, dimethylacetamide, 1,3-dimethyl-2-imidazolidinone, N-methylpyrrolidone, etc.), nitrile solvents (acetonitrile etc.), and the like.
  • ether compounds tetrahydrofuran, anisole, 1,4-dioxane, cyclopentyl methyl ether, etc.
  • alcohols methanol, ethanol, allyl alcohol, etc.
  • ester compounds ethyl acetate etc.
  • nonpolar solvents examples include aliphatic organic solvents, such as pentane, hexane, cyclohexane, and heptane; aromatic solvents, such as benzene, toluene, xylene, and mesitylene; and the like.
  • a fluorescent dye comprising the phosphole compound of the present invention is a highly versatile dye.
  • the protein labeling agent (in particular, antibody labeling agent) of the present invention is preferably in the form of a solution, as described above.
  • a pH of about 5 to 11 is preferable, and a pH of about 6.5 to 7.5 is more preferable.
  • a buffer such as HEPES buffer, tris buffer, tricine-sodium hydroxide buffer, phosphate buffer, or phosphate-buffered physiological saline
  • HEPES buffer such as HEPES buffer, tris buffer, tricine-sodium hydroxide buffer, phosphate buffer, or phosphate-buffered physiological saline
  • the melting point (mp) or decomposition temperature was measured with a Yanaco MP-S3 apparatus.
  • 1 H NMR spectrum, 13 C ⁇ 1 H ⁇ NMR spectrum, and 31 P ⁇ 1 H ⁇ NMR were determined in CDCl 3 or DMSO-d 6 used as a solvent with a JEOL AL-400 ( 1 H: 400 MHz, 13 C: 100 MHz, 31 P: 162 MHz) or JEOL A-600 spectrometer ( 1 H: 600 MHz, 13 C: 150 MHz, 13 P: 243 MHz). Chemical shifts are expressed in 6 ppm.
  • TLC Thin layer chromatography
  • silica gel 60F 254 Merck
  • Column chromatography was performed using a PSQ100B neutral silica gel (produced by Fuji Silysia Chemical Ltd.). Recycling preparative high-performance liquid chromatography (HPLC) was performed using an LC-918 (produced by Japan Analytical Industry Co., Ltd.) equipped with a reverse-phase column (Wakosil-II 5C18 HG Prep), or a YMC LC-forte/R equipped with a reverse-phase column (YMC-DispoPackAT ODS). All reactions were performed in a nitrogen atmosphere unless otherwise specified.
  • Tf represents trifluoromethane sulfonyl
  • TMS represents trimethylsilyl
  • Pd(PPh 3 ) 4 represents tetrakis(triphenylphosphine) palladium (0)
  • Et 3 N represents triethylamine; the same applies below.
  • a solution of 1-bromonaphthalen-2-yl triflate (99.44 g, 280 mmol) and 4-trimethylsilylphenylacetylene (48.80 g, 280 mmol) in triethylamine (Et 3 N; 500 mL) was degassed by bubbling dry nitrogen gas for 20 minutes.
  • the obtained solid was purified by silica gel column chromatography (CH 2 Cl 2 ⁇ 10:1 CH 2 Cl 2 /ethyl acetate) and recrystallization with methanol (MeOH; 100 mL) to obtain 19.48 g of the desired compound 2 as a yellow solid (38.7 mmol, yield: 48%).
  • HSiCl 3 (8.07 mL, 80.0 mmol) was added at once to a suspension of compound 2 obtained in Synthesis Example 2 (10.07 g, 20.0 mmol) in anhydrous toluene (30 mL). After the resulting mixture was stirred at 50° C. for 1 hour, all volatiles were distilled off under reduced pressure. Toluene (20 mL) was then added to the resulting mixture, and the obtained suspension was filtered through Celite (registered trademark). The filtrate was washed with toluene (10 mL). After the filtrate was concentrated under reduced pressure, the obtained white solid was suspended in anhydrous diethyl ether (Et 2 O; 100 mL).
  • Sc(OT f ) 3 represents scandium trifluoromethanesulfonate (III); the same applies below).
  • One of the doublet signals of a quaternary carbon paired with the signal at 138.07 ppm, one of the doublet signals of a CH carbon paired with the signal at 132.45 ppm, and one of the doublet signals of a CH carbon paired with the signal at 121.23 ppm may be overlapped with other signals; 31 P ⁇ 1 H ⁇ NMR (162 MHz, CDCl 3 ): ⁇ 23.73. HRMS (APCI): m/z calcd. for C 42 H 38 O 3 PSi: 649.2322 ([M+H] + ); found. 649.2325.
  • a solid of compound 5 obtained in Synthesis Example 5 (1.405 g, 2.00 mmol), 3-(tert-butoxycarbonyl)phenylboronic acid (0.888 g, 4.00 mmol), tetrakis(triphenylphosphine)palladium (0) (Pd(PPh 3 ) 4 ; 0.116 g, 0.100 mmol) and K 3 PO 4 (2.547 g, 12.0 mmol) were added to a mixed solvent of degassed toluene (24 mL) and H 2 O (6 mL). The resulting mixture was stirred at 80° C. for 12 hours.
  • One of the doublet signals of a quaternary carbon paired with the signal at 133.20 ppm, one of the doublet signals of a quaternary carbon paired with the signal at 132.61 ppm, and one singlet signal of a CH carbon may be overlapped; 31 P ⁇ 1 H ⁇ NMR (162 MHz, CDCl 3 ): ⁇ 23.73. HRMS (APCI): m/z calcd. for C 50 H 42 O 5 P: 753.2764 ([M+H] + ); found. 753.2749.
  • One of the doublet signals of a quaternary carbon paired with the signal at 157.92 ppm, one of the doublet signals of a quaternary carbon paired with the signal at 137.71 ppm, and twenty four singlet signal of an alkyl CH carbon may be overlapped; 31 P ⁇ 1 H ⁇ NMR (243 MHz, CDCl 3 ): ⁇ 23.39.
  • Lithium hydroxide monohydrate (LiOH.H 2 O; 1.00 g, 23.8 mmol) was added to a solution of compound 9 (350 mg, 0.233 mmol) obtained in Example 4 in tetrahydrofuran (THF; 10 mL) and H 2 O (10 mL). After stirring at room temperature for 16 hours, the mixture was acidified with hydrochloric acid (1M, 30 mL). The mixture was extracted with ethyl acetate (EtOAc; 200 mL) twice. The combined organic layer was washed with saturated saline (50 mL) 3 times, then dried over anhydrous Na 2 SO 4 , and filtered.
  • THF tetrahydrofuran
  • EtOAc ethyl acetate
  • NHS N-hydroxysuccinimide
  • DMAP N,N-dimethyl-4-aminopyridine
  • EDCI 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride
  • DMF dimethylformamide
  • One singlet signal of an aromatic quaternary carbon, two doublet signals of an aromatic quaternary carbon, one singlet signal of an aromatic CH carbon, and eighteen singlet signal of an alkyl CH carbon may be overlapped; 31 P ⁇ 1 H ⁇ NMR (243 MHz, CDCl 3 ): ⁇ 23.46.
  • One singlet signal of a quaternary carbon, one of the doublet signals of a quaternary carbon paired with the signal at 133.23 ppm, one of the doublet signals of a quaternary carbon paired with the signal at 132.90 ppm, one of the doublet signals of a quaternary carbon paired with the signal at 131.73 ppm, and three singlet signals of alkyl carbons may be overlapped.
  • N,N,N′,N′-tetramethyl-O—(N-succinimidyl)uronium tetrafluoroborate (TSTU; 9.2 mg, 0.031 enol) was added to a solution of compound 12 obtained in Example 7 (14.0 mg, 0.0153 mmol) and diisopropylethylamine (DIPEA; 0.10 mL, 0.61 mmol) in anhydrous DMSO (1 mL). After stirring at room temperature for 1 hour, 4-aminobutyric acid (10.3 mg, 0.100 mmol) was added to the reaction mixture.
  • DIPEA diisopropylethylamine
  • Triethylamine (Et3N; 28 ⁇ L, 200 ⁇ mol) was added to a solution of Compound 11 obtained in Example 6 (10.0 mg, 9.9 ⁇ mol) and N-(2-aminoethyl)maleimide trifluoroacetate (2.5 mg, 9.8 ⁇ mol) in anhydrous dimethyl sulfoxide (DMSO; 1 mL). After the resulting mixture was stirred at room temperature for 2 hours, the mixture was purified by reverse-phase HPLC (55:45 H 2 O/CH 3 CN, +0.1% trifluoroacetic acid (TFA)) to obtain 3.2 mg of the desired compound 12 (Phox-maleimide) as a yellow solid (3.1 mmol, yield: 32%).
  • DMSO dimethyl sulfoxide
  • Alexa Fluor 488 commercially available, was used as the fluorescent dye of Comparative Example 4.
  • FIG. 1 shows the results.
  • Phox 430 NHS Ester obtained in Example 6 was dissolved in various solvents at a concentration of about 10 ⁇ 5 M. UV-visible absorption and fluorescence spectra, absolute fluorescence quantum yield, fluorescence lifetime, etc. of these solutions were measured. Alexa Fluor 430 obtained in Comparative Example 1 and Atto 425 obtained in Comparative Example 2 were also dissolved in HEPES at a concentration of about 10 ⁇ 5 M. UV-visible absorption and fluorescence spectra, absolute fluorescence quantum yield, fluorescence lifetime, etc. of these solutions were also measured. Table 1 and FIG. 2 show the results.
  • the phoshole compound of the present invention in any of various solvents can fluoresce under light in the visible light region (in particular, at about 400 to 500 nm; the absorption spectral behavior and the fluorescence spectral behavior are almost the same). It can also be understood that the phosphole compound exhibits high brightness even in aqueous solvents.
  • the phosphole compound of the present invention further has a feature that it has a large Stokes shift (5020 cm ⁇ 1 ).
  • the phosphole compound of the present invention has a feature that it has a maximum absolute fluorescence quantum yield of 0.90, which indicates brightness comparable to that of conventional fluorescent dyes, and also has a fluorescence lifetime of 6 ns or more, which is much longer than a conventional lifetime of about 1 to 4 ns.
  • Phox 430 NHS Ester obtained in Example 6 and C-Naphox obtained in Comparative Example 3 were dissolved at a concentration of about 10 ⁇ 6 M in mixed solvents of DMSO and HEPES. Absolute fluorescence quantum yields of these solutions were measured. The measurement was performed in the mixed solvents containing HEPES at various ratios, and fluorescence quantum yield variation relative to water content was evaluated.
  • FIG. 3 show the results. The fluorescence quantum yield is plotted on the ordinate of FIG. 3 ; 100% shows the theoretical upper limit of fluorescence quantum yield. The results clearly show that as compared with C-Naphox of Comparative Example 3, whose fluorescence quantum yield decreased with an increase of water content, the phosphole compound of the present invention can maintain a high fluorescence quantum yield even in solvents containing water.
  • FIGS. 4 and 5 show the results.
  • the results show that photoirradiation of Phox-COOH and C-Naphox for 5 hours caused almost no reduction in fluorescence intensity (99% of the dyes remained intact), whereas only 46.2% of the fluorescence intensity of Alexa Fluor 488 persisted under the photoirradiation for 5 hours, and the photoirradiation reduces the fluorescence intensity of Alexa Fluor 488.
  • the phosphole compound of the present invention has high light resistance comparable to that of C-Naphox, and that its properties are far superior to those of Alexa Fluor 488.
  • the fluorescence spectra of Phox-COOH (10 ⁇ M) obtained in Example 8 were measured in aqueous solutions of various pH values. For adjustment in the range of pH 3 to pH 6, citric acid/Na 2 HPO 4 buffer was used. For adjustment in the range of pH 7 to pH 8, Na 2 HPO 4 /NaH 2 PO 4 buffer was used. For adjustment in the range of pH 9 to pH 11, Na 2 CO 3 /NaHCO 3 buffer was used. The method for measuring the fluorescence spectra was otherwise the same as in Test Example 2.
  • FIG. 6 shows the results. The results show that fluorescence wavelength did not change with pH and that fluorescence intensity was also substantially maintained with minor changes according to the pH (the highest fluorescence intensity being at a pH of 9).
  • Phox-NHS Ester obtained in Example 9 was conjugated to a goat anti-mouse IgG antibody to form a Phox-antibody conjugate.
  • the degree of labeling (DOL) of the sample prepared from 20 ⁇ g of Phox-NHS Ester and 0.50 mg of IgG antibody in 0.25 mL of a labeling buffer (pH of 8.3) was determined to be 2.8.
  • the photophysical properties of Phox-antibody conjugate were almost identical to those of antibody-free Phox-COOH in PBS (pH 7.4), which indicates that there was almost no interaction between the fluorophores or with the amino acid residues of the antibody.
  • the labeling buffer at a pH of 8.3 was prepared by mixing PBS buffer (pH of 7.4) with 0.2M NaHCO 3 at a ratio of 20:1 (v/v).
  • a DMSO (10 ⁇ L) solution of Phox-NHS Ester (Example 9; 0.020 mg) was added to a labeling buffer (pH of 8.3, 0.25 mL) of goat anti-mouse antibody IgG (0.50 mg), and the resulting mixture was cultured at room temperature for 1 hour. After free Phox-NHS Ester was removed by Sephadex G-25 column chromatography, the degree of labeling (DOL) (the number of fluorophores bound per antibody) was measured.
  • DOL degree of labeling
  • the DOL was calculated in accordance with the calculation method described in the “Determination of Degree of Labeling (DOL)” section in the manual of ThermoFisher Scientific's Alexa Fluor (registered trademark) 488 Microscale Protein labeling Kit (https://tools.thermofisher.com/content/sfs/manuals/mp30006.pdf), except that A 280 and A 426 were used in place of A 280 and A 494 for calculation.
  • the DOL calculation method is as follows.
  • ⁇ 280 and ⁇ max represent absorption coefficients of a fluorescent dye at 280 nm and at the absorption maximum (426 nm).
  • a max and A 280 represent absorbance of the Phox-antibody conjugate at the absorption maximum of each dye (426 nm) and 280 nm.
  • the difference between A 280 and A max ⁇ CF 280 refers to the absorbance of the antibody itself (A protein ).
  • ⁇ protein represents the absorption coefficient of the antibody at 280 nm.
  • HeLa cells (RIKEN Cell Bank, Japan) were cultured in Dulbecco's modified Eagle's medium (DMEM, Sigma) containing 10% fetal bovine serum (FBS, Gibco) and 1% antibiotic-antimycotic (AA, Sigma) at 37° C. in a 5% CO 2 /95% air incubator. Three days before imaging, the cells (5 ⁇ 10 4 ) were seeded on a glass-bottom 8-well plate. Immunofluorescently labeled tubulin and vimentin of fixed HeLa cells were prepared in the following manner. 1) HeLa cells were fixed with 4% formaldehyde and cultured at room temperature for 20 minutes.
  • DMEM Dulbecco's modified Eagle's medium
  • FBS fetal bovine serum
  • AA antibiotic-antimycotic
  • Test Example 8 Fluorescent Imaging No. 1
  • a super-resolution TCS SP8 STED microscope equipped with an HCXPL APO 100 ⁇ /1.40 oil immersion lens was used for confocal imaging and STED imaging.
  • confocal imaging cells were irradiated with a 470-nm laser (wavelength-tunable white excitation laser, 80 MHz, output power: 30%, AOTF: 90%), and fluorescent signals within the range of 480 to 585 nm were detected.
  • a wavelength-tunable white excitation laser (470 nm, 80 MHz, output power: 40%, AOTF: 90%) and a CW-STED laser (592 nm, CW laser, output power: 20%, AOTF: 80%) were used with the emission detection window being set at 480 to 585 nm, and the time gating method (time range: 0.5 to 12 ns) was used.
  • a wavelength-tunable white excitation laser (470 nm, 80 MHz, output power: 80%, AOTF: 90%) and a CW-STED laser (a 592-nm CW laser, output power: 20%, AOTF: 80%) were used with the luminescence detection window being set at 480 to 585 nm, and the time gating method (time range: 0.5 to 12 ns) was used.
  • Z-scan STED images were subjected to Huygens deconvolution to construct a 3D structure of tubulin.
  • Huygens deconvolution was performed. These images were analyzed with the ImageJ software (http://imagej.nih.gov/ij/).
  • FIG. 8 shows the results of confocal imaging and STED imaging of vimentin immunofluorescently labeled with Phox-NHS Ester (Example 9).
  • FIG. 8 shows a confocal microscopy image ( a ) and a STED microscopy image ( b ) of tubulin filaments immunolabeled with Phox-NHS Ester (Example 9) in a fixed HeLa cell, and the corresponding optical resolutions ( c ) of the confocal (orange line) and STED (crimson line) microscopy images.
  • FIG. 8( a ) and FIG. 8( b ) include inserts of enlarged images of the selected portions.
  • a wavelength-tunable white excitation laser (470 nm, 80 MHz, output power: 40%, AOTF: 90%) and a CW-STED laser (592-nm CW laser, output power: 20%, AOTF: 80%) were used.
  • Scale bars in FIG. 8 ( a ) and FIG. 8 ( b ) indicate 2 ⁇ m. This result clearly shows that the phosphole compound of the present invention can be used to label a protein (in particular, an antibody) and fluoresce; STED imaging using this phosphole compound can achieve a spatial resolution of more than 200 nm.
  • FIG. 9 shows a comparison of photostability between Alexa Fluor 488 (Comparative Example 4) and Phox-NHS Ester (Example 9) under STED conditions.
  • STED microscopy images of tubulin filaments immunolabeled with Alexa Fluor 488 (Comparative Example 4; a) and Phox-NHS Ester (Example 9; b) were repeatedly captured 5 times consecutively.
  • changes in fluorescence intensity versus the number of repetition of STED imaging were plotted in FIG. 9( c ) .
  • FIG. 9( c ) shows to what degrees the fluorescence intensity can be maintained from the initial value.
  • FIG. 10 and FIG. 11 show a comparison between Alexa Fluor 488 (Comparative Example 4) and Phox-NHS Ester (Example 9) in photostability during Z-scan STED imaging.
  • FIG. 10( a ) and FIG. 11( a ) show confocal fluorescence microscopy images with Alexa Fluor 488 (Comparative Example 4) and those with Phox-NHS Ester (Example 9).
  • FIG. 10( b ) and FIG. 11( b ) show Z-scan STED microscopy images in a depth of 2 ⁇ m. The Z-scanning step was set to intervals of 200 nm, and 11 slides were recorded. After analysis of the 11 slides and image reconstruction, the three-dimensional structure of tubulin filaments was obtained with a dimension of 11.62 ⁇ 11.62 ⁇ 2.00 ⁇ m 3 , as shown in FIG. 10( c ) and FIG. 11( c ) .
  • a wavelength-tunable white excitation laser (470 nm, 80 MHz, output power: 40%, AOTF: 90%) and a CW-STED laser (592-nm CW laser) output power: 20%, AOTF: 80%) were used.
  • Tubulin filaments immunolabeled with Phox-NHS Ester (Example 9) and those with Alexa Fluor 488 (Comparative Example 4) were both imaged under the same conditions.
  • Scale bars in FIG. 10 ( a ) and FIG. 11 ( a ) indicate 2 ⁇ m.
  • Imaging experiments were performed using a Leica TCS SP8 STED 3X system (Leica Microsystems), including an inverted DMI6000 CS microscope equipped with a tunable (470 to 670 nm) pulsed white-light laser (WLL; pulse repetition rate of 78 MHz) for excitation and a STED laser (continuous wave at 592 nm) for depletion.
  • WLL pulsed white-light laser
  • STED laser continuous wave at 592 nm
  • confocal imaging and STED imaging a HyD detector and a 100 ⁇ oil immersion objective lens (NA: 1.4) were used.
  • the dyes were excited with the WLL at 470 nm, and fluorescent signals were collected between 480 nm and 585 nm with a time gating interval of 0.5 to 12 ns.
  • Z-stack images were obtained with increments of 50 nm.
  • the images were first deconvoluted using the Huygens Deconvolution software (Scientific Volume Imaging), and the deconverted images were further processed with the ImageJ image analysis software (http://imagej.nih.gov/ij/).
  • ImageJ image analysis software http://imagej.nih.gov/ij/.
  • the total signal strength of each image was normalized to the value of the first image and plotted as a function of the number of recorded confocal images.
  • the decay curve was analyzed as a pseudo first-order reaction (Dyes Pigm. 1998, 37, 213-222), and the photobleaching rate constant was calculated from the slope of the straight line. Relative photostability to Alexa Fluor 430 was determined by using the inverse of the photobleaching rate constant.
  • z-scan STED imaging of microtubules was performed.
  • the construction of a three-dimensional (3-D) image from two-dimensional STED images is challenging because rapid photobleaching during sequential xy-scans in usually unavoidable when using conventional dyes.
  • the immunolabeled microtubules around the nucleus of the cell were scanned along the z-axis with a step of 50 nm.
  • sufficient brightness and super-resolution of the microtubules were maintained while recording the STED images from the bottom to the top of the cell (z-depth of 4.0 ⁇ m).
  • Alexa Fluor 430 In contrast, almost no fluorescence signals were retained after 10 images when Alexa Fluor 430 and STAR 440SX were used.
  • Alexa Fluor 488 and Atto 425 have slightly better photostability, and the bleaching rates of these dyes were 2.3 and 5.3 times, respectively, slower than that of Alexa Fluor 430 (Table 2). Because Alexa Fluor 430 showed the largest difference in photostability, Alexa Fluor 430 was decided to be used for comparison with Phox-COOH (PB430).
  • Tubulin and vimentin of the fixed cells were then stained with Phox-COOH (PB430)-conjugated secondary antibodies and Alexa Fluor 430-conjugated secondary antibodies, respectively.
  • the first STED image was recorded ( FIG. 19( a ) ), which should include both cytoskeletons.
  • the second image was recorded under identical conditions. This resulted in the disappearance of several filament structures ( FIG. 19( b ) ).
  • Subtracting (removing) the second image ( b ) from the first image ( a ) enables obtaining a STED image of the lost filaments, which corresponds to the Alexa Fluor 430-labeled vimentin filaments ( FIG. 19( c ) ).

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